Nucleolin (also known as P92 and C23) is the most abundant nucleolar phosphoprotein in actively growing cells (Srivastava et al., FEBS Lett., 1989, 250, 99-105; Srivastava et al., J. Biol. Chem., 1990, 265, 14922-14931). It has been described by several groups and shown to participate primarily in ribosome biogenesis (Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114; Sipos and Olson, Biochem. Biophys. Res. Commun., 1991, 177, 673-678) and transport of ribosomal components (Schmidt-Zachmann et al., Cell, 1993, 74, 493-504). Nucleolin contributes to ribosome biosynthesis by transiently binding to the pre-ribosomes in the nucleolus via a ribonucleoprotein consensus sequence (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925; Ghisolfi-Nieto et al., J. Mol. Biol., 1996, 260, 34-53; Sapp et al., Eur. J. Biochem., 1989, 179, 541-548). Here, nucleolin can represent up to 5% of the total nucleolar protein (Lapeyre et al., Proc. Natl. Acad. Sci. U.S.A., 1987, 84, 1472-1476; Sapp et al., Eur. J. Biochem., 1989, 179, 541-548). However, it has also been shown to be involved in cytokinesis, nucleogenesis, cell proliferation and growth, transcriptional repression, replication, signal transduction and chromatin decondensation reviewed in (Tuteja and Tuteja, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 407-436).
The multifunctionality of this protein arises from the presence of distinct structural and functional domains within the protein (Creancier et al., Mol. Biol. Cell., 1993, 4, 1239-1250; Sapp et al., Eur. J. Biochem., 1989, 179, 541-548). Three domains have been described within the nucleolin protein, the N-terminal domain, the central domain and the C-terminal domain. Contained in the N-terminal domain are sequences that show homology with the high-mobility group (HMG) and are responsible for interactions with chromatin (Erard et al., Eur. J. Biochem., 1988, 175, 525-530). The central domain contains four RNA recognition motifs and binds specifically with the short stem loop of the 18S and 28S ribosomal RNA (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925) while the C-terminal domain contains regions that are capable of unstacking bases in RNA (Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114; Ghisolfi-Nieto et al., J. Mol. Biol., 1996, 260, 34-53). Nucleolin contains a bipartite nuclear localization signal, spanning both the N-terminal and central regions of the protein, which facilitates transport into the nucleus where nucleolin accumulates due to interactions with other proteins (Schmidt-Zachmann and Nigg, J. Cell Sci., 1993, 105, 799-806).
The domain structure of nucleolin has led the protein to be classified as an Ag-NOR protein (Active ribosomal gene located in the Nucleolar Organizer Region) otherwise known as markers of active ribosomal genes (Roussel et al., Exp. Cell. Res., 1992, 203, 259-269). It has been shown that transcription of ribosomal genes requires the presence of Ag-NOR proteins and the expression of Ag-NOR proteins has been associated with the prediction of tumor growth rate in cancers.
Nucleolin has also been purified as a matrix attachment region (MAR) binding protein from human erythroleukemia cells. In these studies, nucleolin was shown to participate in the anchoring of chromatin loops to the nuclear matrix (Dickinson and Kohwi-Shigematsu, Mol. Cell. Biol., 1995, 15, 456-465).
Nucleolin is highly phosphorylated and has been shown to be a substrate for casein kinase II (Csermely et al., J. Biol. Chem., 1993, 268, 9747-9752; Schneider and Issinger, Biochem. Biophys. Res. Commun., 1988, 156, 1390-1397), Protein kinase C-.xi. (Zhou et al., J. Biol. Chem., 1997, 272, 31130-31137), and Cdc2 (Belenguer et al., Mol. Cell. Biol., 1990, 10, 3607-3618). Furthermore, the phosphorylation of nucleolin has been shown to regulate the subcellular localization of the protein.
Nucleolin also undergoes self-cleavage, which is decreased when cells enter a proliferative stage, as well as being cleaved by Granzyme A, an esterase secreted by cytotoxic lymphocytes (Chen et al., J. Biol. Chem., 1991, 266, 7754-7758; Fang and Yeh, Exp. Cell. Res., 1993, 208, 48-53; Pasternack et al., J. Biol. Chem., 1991, 266, 14703-14708). The cleavage and concomitant degradation of the protein provides for post-translational regulation of nucleolin.
Anti-nucleolin antibodies have been found in the sera of patients with systemic connective tissue diseases including systemic lupus erythromatosus (SLE) (Minota et al., J. Immunol., 1990, 144, 1263-1269; Minota et al., J. Immunol., 1991, 146, 2249-2252) and scleroderma-like chronic graft vs. host disease (Bell et al., Br. J. Dermatol., 1996, 134, 848-854). The pharmacological modulation of nucleolin expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of nucleolin. Consequently, there remains a long felt need for agents capable of effectively inhibiting nucleolin function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of nucleolin expression.
The present invention provides compositions and methods for modulating nucleolin expression.